1. Field of the Invention
The invention concerns the fields of molecular biology, cell biology, clinical medicine, pharmacotherapy, and oncology.
2. Description of Related Art
Transcriptional enhancer factor 1-related (RTEF-1) gene is a member of the TEA DNA binding domain gene family. The TEA DNA binding domain gene family is highly conserved from Aspergillus nidulans, yeast, Drosophila, mice to human. The TEA DNA binding family of proteins can be involved in both activation and repression of different genes and their particular function can be modified by association with other proteins (Kaneko & DePamphilis, 1998). Expression of specific members of these genes has been identified in various mammalian tissues, including heart, skeletal muscle, pancreas, placenta, brain and lung (Stewart et al., 1996; Yasunami et al., 1996; Farrance et al., 1996). Isoforms arising from alternative splicing of mRNA from a single gene, for transcriptional enhancer factor-1 (TEF-1) have been identified within a single tissue such as the pancreas (Zuzarte et al., 2000; Jiang et al., 2000). The expression profile of these genes within the mammalian eye has not been reported.
Transcripts of the RTEF-1 gene were first identified in chicken tissue and demonstrated to be enriched in cardiac and skeletal muscle (Farrance et al., 1996). The chicken RTEF-1 binds to the myocyte-specific CAT (M-CAT) cis DNA elements and regulates expression of muscle specific genes, and requires muscle specific cofactors for full transcriptional activation. Random screening of 2166 human colorectal cancer cDNA library identified a partial cDNA RTEF-1 sequence which lead to the isolation of a full length human homolog of the avian RTEF-1 from a heart cDNA library (Stewart et al., 1996; Frigerio et al., 1995). Northern blot analysis of human tissue indicated highest levels of expression in skeletal muscle and pancreas, with lower levels in heart, kidney and placenta, whereas message was not detected in liver, lung or brain (Stewart et al., 1996). Northern blot analysis of the mouse homolog of RTEF-1 indicates a different tissue expression pattern when compared to human. Adult mouse lung tissue expressed the highest level, with very low levels in kidney, heart and skeletal muscle and undetectable amounts in liver, thymus, spleen and brain, whereas RTEF-1 message was abundant in mouse embryonic skeletal muscle (Yockey et al., 1996). An alternatively spliced mouse isoform of RTEF-1 that lacks exon 5 when compare to the full length gene has been identified in mouse skeletal muscle cells (Yockey et al., 1996).
RTEF-1/TEAD4 has been shown to bind with Yes-associated protein (YAP) and modulate gene expression (Vassilev et al., 2001). Interacting at the end of the Hippo Pathway, TEAD and YAP control organ size during development and, thus, play an important role in the coordination of cell growth, proliferation, and apoptosis (Ota and Sasaki, 2008). Studies have shown that cell to cell interactions elicit signals though the Hippo Pathway which govern YAP-dependent RTEF-1/TEAD4 transcriptional activity (Nishioka et al., 2009). Disruption of the Hippo Pathway or altered activity of YAP (expression or localization) can lead to cell overgrowth and survival (Zeng and Hong, 2008). The four human TEAD proteins share more than 72% homology. Variable regions are found in the proline-rich domain and the n-terminus. The n-terminus is thought to be a target for phosphorylation whereas the c-terminus has been shown to bind with YAP. YAP binds to all four TEAD proteins (Vassilev et al., 2001).
YAP is found to be over expressed and diffuse in many tumors. YAP location and degree of overexpression varies between cancer types (Steinhardt et al., 2008). Wnt and Akt/PKB are two other pathways that are tightly regulated during development and seem to play large roles in tumor malignancy. Reduction of over expressed YAP activity is thought to be correlated with decreased cell migration, decreased Akt activation, and increased E-cadherin levels. Studies have shown that 50-80% of metastatic cancers express less E-cadherin compared to normal tissues (Orsulic et al., 1999) and YAP overexpression is found in many highly metastatic cancers that are associated with short survival (Wang et al., 2010). Immunoblotting suggests that E-cadherin and occludin levels decrease while N-cadherin, fibronectin, and Akt phosphorylation increase in the presence of YAP (Overholtzer et al., 2006). Likewise, YAP S94A mutation (abolishes YAP and TEAD4 interaction) results in an increase in E-cadherin and gamma-Catenin expression while N-cadherin and fibronectin level decrease (Zhao et al., 2008). Some studies have shown that tumor cells will revert to a benign phenotype upon E-cadherin re-establishment. E-cadherin/B-catenin complex is essential for cell adhesion. A decrease in E-cadherin results in an increase of free B-catenin, which can enter the nucleus and activate target genes which leads to cancer (Wnt pathway) (Semb and Christofori, 1998).
Balancing cell proliferation and apoptosis is essential for proper tissue growth, development, and function. Disruption can lead to excessive tissue loss with subsequence loss of function as in the case of excessive apoptosis or uncontrolled cell proliferation. The Hippo pathway is a potent regulator of tissue homeostasis by controlling cell growth, division, and apoptosis. The potent effect of YAP on cell growth, division, and apoptosis supports the notion that YAP functions as to maintain tissue homeostasis. Once dysregulated, it can lead to a malignant phenotype. Malignant cells might produce excess YAP during genomic amplification that might overwhelm the normal physiologic regulatory systems and result in abnormal cytoplasmic accumulation. Accumulation of YAP within the cytoplasm maintains a constant pool of the protein for nuclear translocation. The stability of YAP may be altered in neoplastic tissues resulting in ineffective protein turnover and excessive YAP activity.
Vascular endothelial growth factor (VEGF) is one pro-angiogenic factor that is known to be up regulated in retinal tissue under hypoxic conditions (Young et al., 1997; Pierce et al., 1996; Donahue et al., 1996; Pe'er et al., 1995). Recently the full length RTEF-1 protein has been identified to not only bind to the VEGF promoter but also to up-regulate the expression of VEGF, for instance under hypoxic conditions in bovine aortic endothelial cells (BAEC) (Shie et al., 2004). Microarray analysis revealed that RTEF-1 expression was up-regulated by 3-fold in BAEC under hypoxic conditions. Surprisingly, RTEF-1 mediated VEGF gene activation via interaction with Sp 1 elements within the VEGF promoter and not M-CAT motifs. In addition RTEF mediated expression of VEGF is achieved independently of the hypoxia-inducible factor (HIF-1) and hypoxia responsive element (HRE) pathway of activation (Shie et al., 2004).
VEGF over-expression has been implicated in a variety of angiogenic disorders such as tumor angiogenesis and aberrant neovascularization. For example, it is well established that VEGF plays an important role in the development and severity of retinopathy of prematurity (ROP) and other ocular neovascular diseases (Lashkari et al., 2000; Miller, 1997; Vannay et al., 2005; Young et al., 1997). Given the prominent role of VEGF in such disorders a number of therapeutic strategies for inhibiting VEGF activity have been developed. However, current VEGF blockade therapies typically involve inhibiting the interaction of extra cellular VEGF with cognate cell surface receptors. Thus, there is a need for alternative strategies for VEGF blockade such as method for inhibiting VEGF production.